Delivery of a BET protein degrader via a CEACAM6-targeted antibody–drug conjugate inhibits tumour growth in pancreatic cancer models

Pancreatic ductal adenocarcinoma (PDAC) has the worst prognosis of all cancers. To improve PDAC therapy, we establish screening systems based on organoid and co-culture technologies and find a payload of antibody–drug conjugate (ADC), a bromodomain and extra-terminal (BET) protein degrader named EBET. We select CEACAM6/CD66c as an ADC target and developed an antibody, #84.7, with minimal reactivity to CEACAM6-expressing normal cells. EBET-conjugated #84.7 (84-EBET) has lethal effects on various PDAC organoids and bystander efficacy on CEACAM6-negative PDAC cells and cancer-associated fibroblasts. In mouse studies, a single injection of 84-EBET induces marked tumor regression in various PDAC-patient-derived xenografts, with a decrease in the inflammatory phenotype of stromal cells and without significant body weight loss. Combination with standard chemotherapy or PD-1 antibody induces more profound and sustained regression without toxicity enhancement. Our preclinical evidence demonstrates potential efficacy by delivering BET protein degrader to PDAC and its microenvironment via CEACAM6-targeted ADC.

The language of 'potential curability' should be removed from the manuscript.While the data in mice look promising, preclinical studies are premature for discussions on curability, particularly given the daunting challenges associated with pancreatic cancer.

Specific comments:
Page 4: PC-3 is a common prostate cancer cell line, so it may be helpful to explicitly state this is a different (pancreatic) line, since this proprietary model would be less commonly known.
Page 5 and 6: PNU, calicheamicin, and PBD agents have pM potency, and some have bystander effects.Why were the IC50 values thousands of times lower?Are cancer cells dying but fibroblasts surviving?Is there another explanation?Page 8: What is the expression level of CEACAM6 (receptors/cell)?IHC staining and H-scores are not very informative for quantifying the expression level.For instance, 3+ IHC staining can be 1 million receptors per cell or 6 thousand receptors per cell (e.g.Sharma et al. 2017, Cancer Res).
Page 9: What is the mechanism behind the specificity?Is it related to the affinity?Glycosylation?Have the authors examined the epitope on the antigen?Page 13: It is hard to say if there's no toxicity enhancement, since body weight is a crude indicator.A more nuanced statement would be more appropriate.
Figure 2 -There is little quantification in these images, making it difficult to determine the level of differences.Is there any way to quantify these results? Figure 2d -How is internalization assessed in this figure?While internalization can occur at 37C, the same signal is generated, so it could be surface binding as well.
What is the membrane permeability of the degrader (e.g.PAMPA)?These larger BRo5 compounds are often more challenging to get into/out of cells.
Reviewer #2: Remarks to the Author: Nakazawa and colleagues identify and test a novel ADC for the treatment of pancreatic cancer.The manuscript represents a comprehensive preclinical workup of a new therapeutic strategy.The authors identify that a bromodomain and extra-terminal (BET) degrader, termed EBET is effective as a payload for an ADC and then choose CEACAM6 as the target for delivery.The authors develop a novel monoclonal antibody #84.7 specific to CEACAM6.#84.7 appears to have selectivity to CEACAM6 expressed on tumor cells and localize marginally to endogenous CAECAM6 on lung epithelial cells in monkeys.The ADC has impressive single agent activity in PDX models of pancreatic cancer.
There are multiple noteworthy results.1. ID of EBET as a therapeutic modality using an organoid screen; 2. Demonstration that EBET alters fibroblast activity (reporter assays) in a co-culture system and reduces induction of a stem-like state in tumor cells in vitro; 3. Development of a novel mAb specific for CEACAM6 and characterization of the mAb, including showing limited activity of the ADC in an ALI model and in vivo localization studies in monkeys, which helps validate the concept of targeting CEACAM; 4. Demonstration of in vivo efficacy in PDX models that represent classical or basal PDA; 5. Data suggestive of promising activity of the 84.7-EBETADC in combination with anti-PD-1 in an engineered model.
The work provides compelling evidence that 84.7-EBET ADC is an attractive therapeutic that is poised for translation to the clinic.The work is original.
The methods are adequately described and the study encompasses a wide breadth of techniques, results of which in general support the overall conclusions of the study.

Comments:
1.A challenge when developing human specific reagents is the limited toxicity of the construct because normal cells in the mouse are not bound by the therapeutic.The authors provide evidence that 84.7-EBET will be well tolerated in humans (ALI model, in vivo localization in monkeys, no weight loss in mice); however the lack of binding of 84.7 to mouse CEACAM6 complicates interpretation of any toxicity.I suggest that the authors 1. report or determine the MTD of EBET alone in mice; 2. Repeat the ALI study using 84.7-EBET instead of the PNU payload; 3. Determine if 84.7-EBET and KOR-EBET show cytotoxic activity on human PBMCs.3.For all in vivo studies the average size of tumors at the start of therapy should be reported.

In Fig 3c is the viability of CAFs reduced by treatment with any of the ADCs?
Suggested edits: A general edit for clarity is encouraged.
Reviewer #3: Remarks to the Author: In this manuscript, Nakazawa, Miyano and colleagues engineer a novel ADC using a novel BET protein degrader (EBET) conjugated to a newly developed antibody to CEACAM6.They test the novel degrader on both PDAC organoids and bystander CAFs, while the CEACAM6 antibody is tested in monkey tissues and used against transplanted organoids in mice.The authors also leverage a human co-culture system that brings organoids, CAFS and PBMCs together.Finally, they test the ADC in tumor transplant models and observe dramatic response when they combine the ADC with PD-1 checkpoint immunotherapy.
The novelty of the manuscript is high as a novel drug and antibody are described.The robustness of the in vitro studies is also high as the authors test a large cohort of patient-derived organoids.The mechanisms of action of the ADC is investigated and seem to indicate that bystander effect on CAFs is important.The in vivo validation, while striking, may be problematic as CEACAM6 is not present in mouse tissues and therefore the results may be confounded.As a result, the conclusions may be overstated.
The manuscript could benefit from a careful review as details appear to be missing and figure descriptions are not always accurate.As presented the manuscript is very confusing.
Major comments: 1. "curability" is a strong term considering that the in vivo models used do not really represent the human disease.Mouse cells do not express the CEACAM6 antigen and therefore the tumor cells expressing CEACAM6 make a perfect target for the ADC. 2. The introduction is brief.No discussion is provided for the history of BET inhibitors in PDAC.It would be helpful to introduce these for the readers that may not be familiar.Also, on pages 16-17 there is a whole paragraph dedicated to introducing chemotherapy regimens in PDAC.This could be in the introduction rather than discussion.3. CEACAM6 is not present in mouse cells, therefore using this as the target for the ADC would bias the binding to human tumor cells in PDX models.This is not really discussed.How do the authors plan on addressing this issue in future pre-clinical trials?4. The clustering shown in FIGS1 for activated vs normal stroma is not very convincing.Color legend is missing from the figure or legend.Why are there fewer samples in the MYC signature heatmap? 5. FIGS1b: having the two PDX models on two separate graphs makes comparison of response very challenging especially since the scales of the graphs are different.Please re-graph.6.The call outs in the text for FigS2 a and b seem to be in the wrong order compared to the figure.7. "All reporter activities on LSCs were enhanced by LSC co-culture with PC-3 cancer cells (Fig. 1b)" this is not what figure 1b shows.How do the authors make this conclusion?8. How was this done: "We therefore selected EBET-1055 as a seed compound, optimized it as a payload, and finally obtained the lead payload EBET-1593 (Extended Data Fig. 3b)."There are no details provided in the methods or results.9. How does 84-EBET affect CAF viability in a mono and co-culture in vitro assay?10.The single cell data is poorly described and lacks context.As written, I am not sure if anything new was learned.Were the mice treated for the single cell experiment?11.The tiple co-culture experiments were not autologous, can the authors expand on how meaningful the immune reaction is and much of this phenotype would be translatable to patients where checkpoint inhibition often has no impact on tumor cells?Would using a mouse syngeneic co culture system expressing CEACAM6 be more useful since the authors have access to such a model?12. Can the authors show representative images of the data quantified in figure 6C (this can be added to supplemental figures).In relationship to this experiment, have the authors also checked markers of proliferation and cell death in these mice?Is it possible that the immune reaction observed is due to the overexpression of CEACAM6 on the tumor cells, thus providing a tumorspecific antigen?How would this translate to patients?Finally, it would be beneficial to break down the BRD4+ staining by cell type (tumor vs CAF staining) to demonstrate in this curative model that there is a bystander effect on the CAF BRD4 expression.Similarly staining the tissue with p-STAT3 and a CAF marker would help support the model proposed by the authors.13.Can the authors discuss why their novel ADC outperforms BRD2/4 inhibitors in PDAC?Would other epigenetic targeted drugs perform better in PDAC if they were conjugated to a tumor specific antibody?
Minor issues: 1. Line numbers would make reviewing easier.2. In the introduction, second paragraph, the "#84.7" is repeated twice in a row.3. Please clarify if organoids were directly made from PDX tumors and used immediately for assays or if they were passaged in culture first.
Reviewer #4: Remarks to the Author: Authors of this manuscript reported the development of antibody-BET (bromodomain and Extra-Terminal domain) degrader conjugated molecules, represented by 84-EBET, that delivered the payload (BET degrader) to pancreatic cancer cells overexpressing CEACAM6.In addition, the BET degrader payload (such as EBET-1055) was shown to attenuate proinflammatory and pro-survival genes in tumor-associated fibroblasts that support pancreatic tumor growth.Attenuation of the tumor microenvironment by EBET-1055 would be particularly beneficial for pancreatic cancers because of the dense fibrotic environment surrounding pancreatic cancers seen in vivo models and in clinics.As a single agent, 84-EBET dose-dependently suppressed the tumor growth in xenograft mouse models transplanted with four different pancreatic cancer cells expressing high levels of CEACAM6 in vitro.Interestingly, 84-EBET was also effective in mouse xenograft models transplanted with two of four additional pancreatic cancer cells expressing low levels of CEACAM6.84-EBET as a single agent was shown to be significantly more effective than gemcitabine (current standard therapy for pancreatic cancers) or HEL-EBET (non-targeting payload control) in PC-3 orthotopic transplanted model.Although 84-EBET reduced a set of pro-fibrotic cytokines expression in the tumor and the tumor-associated fibroblasts, 84-EBET synergized more effectively with PD-1 monoclonal antibody in the Pan02/hCECAM6 mouse model without causing toxicity.In summary, the authors first developed a novel class of BET inhibitors that were used to generate BET degrader based on the Proteolysis-targeting chimera (PROTAC) technology.The BET degrader molecules were subsequently coupled to an in-house developed antibody recognizing the pancreatic cancer cells specifically expressed surface marker (CEACAM6) for delivery.By recognizing the BET degrader's utility in suppressing pro-growth inflammatory genes, the authors characterized that 84-EBET suppressed the pro-inflammatory environment at the tumor site via the bystander effect.Finally, 84-EBET can synergize with the immune checkpoint inhibitor (PD1-mAb) to induce a sustained anti-tumor activity in the orthotopic pancreatic cancer cell line mouse model.Overall, this is a comprehensive study that integrates several state-of-the-art drug development technologies to advance the therapeutic development for pancreatic cancers, one of the leading lethal cancers lacking effective therapy.Although the study design was straightforward, I have the following points for the authors to clarify.
1. Chemistry for preparing the new EBET compounds needs to be either provided or referenced.2. PC-3 name has been widely used for another prostate cancer cell line.I suggested the authors use a different name to avoid confusion.Is this cell line the same as the BxPC4 from the ATCC or a different cell line?Also, are PC-3 and PC-42 PDX cell lines?3. How 84-EBET was generated from EBET-1593 and the #84.7 antibody was not clearly described.EBET-15935 did not appear to have many functional groups for conjugation to a second antibody.Was is the mechanism of the release of EBET-1593 payload from 84-EBET after internalization?Some descriptions of the generation of 84-EBET are needed.4. What are the blue and red curves in Fig. 3a?Different compounds? 5.In Fig. 3c, was the non-targeting HEL-EBET expected to be internalized?HEL-EBET appeared to dose-dependent reduction of IL-6.6.A previous study reported that JQ1 inhibited the growth of a subset of pancreatic cancer cell lines grown in 3-dimensional collagen (Mol.Can. Ther., 2014, 13, 1907).In Extended Fig. 3, JQ1 and dBET6 showed poor activity against PC-3 but EBET-590 was more effective.What are the binding affinities differences between JQ1 and EBET-590 against the BET proteins?Was the thousand-fold difference between JQ1 and EBET-590 due to their differences in the binding affinities? 7.In Extended Fig. 4, the degradation of BRD2 and BRD4 by EBET-1055 was shown.What was the effect of EBET-1055 on BRD3 levels?8. PC-42 is a basal-like cell line and CEACAM6low.Why #84.7 showed stronger internalization activity in PC-42 (page 9)? 9.In Fig. 4, a payload with either CEACAM6 targeting or non-targeting (HEL) was used in the mouse study.Was EBET-1593 alone effective in some of these cell lines?In relation to point 5, was HEL-EBET internalized or did HEL conjugation prevent the HEL-EBET to be internalized?The little antitumor effects of HEL-EBET implied that it may not be internalized in cells.10.Any insight on why CEACAM6low PC-3 and KYK-036 were sensitive to the CEACAM6 targeting 84-EBET single agent in Fig. 4? 11.In Fig. 6c BRD4+, two groups were found in the combo treatment at day 7. Can the authors check if the significance were **** relative to the vehicle?12.The data from the combination of 84-EBET and PD-1-mAb was encouraging and promising.Although increased antigen presentation by PROTAC molecules may increase the recognition of the cancer cells by activated T cells (J of immunology, 2021, 207, 493), BET protein inhibition suppressed PD-L1 expression in triple-negative breast cancer cells (Cancer Lett, 2019, 265, 45) and PD-1 expression in T cells (Cell Death & Disease, 2022, 13, 671).In the context of pancreatic cancers, was there any data indicating the impact of the degradation of BET proteins (or BET inhibitors) on the PD-L1/PD-1 expression on the pancreatic cancers or the cell lines used in this study?In addition, cytotoxic T-cell activation is required to sustain the long-lasting efficacy of immune checkpoint inhibitors (PD-1-mAb).Fig. 6 showed that 84-EBET treatment reduced the CD4/8/GzmB+ positive area.What are the impacts of the BET degradation by 84-EBET on CD8+ T cell activation?13.In Fig. 6, no tumor volume difference was found between 84-EBET and combo treatment up to day 20.This is also reflected in little changes in the population of CD4 and CD8 positive or GzmB positive cells between 83-EBET and combo at days 3 and 7. Dramatic tumor volume reduction in the combo treatment group however occurred at around day 24 (Fig. 6b) but not in the PD-1-mAB treatment group.Do the authors have any interpretation of the dramatic effect of the combo treatment on the tumor volumes at a later timepoint?Was it mediated by the reduction of caf to allow T cell infiltration?Why the CD4 and CD8 staining in the tumor area were not done after day 20 that may help explain the treatment outcome?14.Do the authors have the data regarding the half-life of 84-EBET in mice?
Minor: 1. Page 4, should be "complex interaction between cancer cells and stromal.." 2. Page 7, improve the immunosuppressive environment will favor the tumor growth.Is this what the authors meant? 3. Page 30, Real-time BD1 degradation assay.Should be "HEK293/LgBiT/BD1-HiBiT".4. What are the blue curves and dots in Extended Fig. 3c, 3d? Different compounds in the library?
The authors and company have invested a significant amount of effort on the screening and development of an anti-CEACAM6 with BET protein degrader payload antibody drug conjugate for the treatment of pancreatic cancer.Overall, this is an extensive amount of data using a rational screening approach for both a unique payload with cancer cell and stromal cell effects (known to be important in pancreatic cancer) and antibody with high target expression and modest healthy tissue binding (where some may be inaccessible due to the target location).The authors developed the novel ADC, tested it in several different PDX models of pancreatic cancer, examined antibody binding in cyno, tested the biological impact on CAFs via bystander effects in vivo, and tested combination therapy with the ADC.This work is informative to the community developing novel ADCs.There are a few areas to be addressed listed below.

Overall comments:
1.The biggest weakness in the development of this compound is the unknown tolerability of the novel payload.Do the authors have an estimate as to the potential clinical tolerability (e.g.cyno tox data, or related compounds)?Colombo et al.

2022, Cancer
Cell showed that ADCs often don't increase the tolerability of the payload.Likewise, Nessler et al. 2021, Trends in Pharm Sci have highlighted how sufficient antibody dosing is needed for adequate tumor uptake even with bystander payloads.Without an estimate for the tolerability of the ADC, the potential for clinical translation remains uncertain.This is not to say that the data are not promising.It is simply uncertain whether these results will translate to the clinic without an estimate of tolerability.
➢ This manuscript is a report of lead CEACAM6-EBET-ADC.After evaluation of the lead ADC, final optimization of linker payload has been conducted especially focusing assays with human lung epithelium, hematopoietic progenitor cells and myeloid progenitor cells to improve the tolerability.The final ADC candidate did not show any effect on their viability at 3.3 nM but showed lethal effect in PDAC cells at 0.1 nM.The ADC is currently in tolerability testing with dogs and monkeys for clinical trial.Due to our development policies, we are unable to show the structure of the final candidate here but added the data of in vitro efficacy and toxicity and the explanation (Extended Data Fig. 13a-c, lines 2-6 on page 18).
2. Related to the tolerability, what is the expression level on lung and MPCs (receptors/cell)?This will likely determine if a MTD is target-mediated or not.It appears like expression is higher on the lung than the PC-42 cells, although it may not be as accessible as the authors mention.Lung tox has been an issue for multiple ADCs, however.

➢ Human MPCs and lung epithelial cells (HSAEC) showed relatively lower expression of CEACAM6 in vitro
(13,210/MPC and 384,704/HSAEC), while PDAC cells showed higher expression ranging from 393,904/cell to 1,753,688/cell.As noted above, we believe we have achieved a certain amount of therapeutic window considering in vitro efficacy and tolerability assays of the final candidate.We added the data quantifying the number of CEACAM6 molecules on the cell membrane (Extended Data Fig. 6b).
3. What is the linker release mechanism of the ADC?Is it protease cleavable, non-cleavable, etc? Is there a self-immolating portion such that the EBET-1593 is the released compound?This information appears to be missing from the manuscript.
➢ In this manuscript, we are using cathepsin-B-cleavable GGFG linker.EBET-1593 is released after linker cleavage and self-immolation of aminomethylene moiety.We added the explanation in the manuscript (lines 18-19 on page 7, lines 3-5 on page 26).
4. The language of 'potential curability' should be removed from the manuscript.While the data in mice look promising, preclinical studies are premature for discussions on curability, particularly given the daunting challenges associated with pancreatic cancer.
➢ We changed the words to "potential efficacy" (line 11 on page 2, line 1 on page 19).

Specific comments:
1. Page 4: PC-3 is a common prostate cancer cell line, so it may be helpful to explicitly state this is a different (pancreatic) line, since this proprietary model would be less commonly known.
➢ We added the explanation (line 11 on page 5).
2. Page 5 and 6: PNU, calicheamicin, and PBD agents have pM potency, and some have bystander effects.Why were the IC50 values thousands of times lower?Are cancer cells dying but fibroblasts surviving?Is there another explanation?
➢ Some drugs show lower efficacy in 3D culture systems than in 2D culture, and this is often due to the drug's ability to penetrate the tissue.Our bystander assay of ADCs showed EBET compounds had higher cell permeability compared to DNA-binding reagents.PROTACs induce protein degradation through a catalytic process and are constantly recycled, while DNA-binding reagents require covalent or strong binding to DNA for their efficacy.This might contribute the higher efficacy of EBET in organoid models.

Page 8:
What is the expression level of CEACAM6 (receptors/cell)?IHC staining and H-scores are not very informative for quantifying the expression level.For instance, 3+ IHC staining can be 1 million receptors per cell or 6 thousand receptors per cell (e.g.Sharma et al. 2017, Cancer Res).
➢ We added the data quantifying the number of CEACAM6 molecules on the cell membrane (Extended Data Fig. 6b).
Establishment of scoring method of CEACAM6 expression is ongoing for clinical studies.
4. Page 9: What is the mechanism behind the specificity?Is it related to the affinity?Glycosylation?Have the authors examined the epitope on the antigen?➢ We already identified the epitope of our mAb using non-glycosylated CEACAM6.We are continuing our research assuming that different glycosylation pattern on CEACAM6 can alter the recognition by #84.7.The data will be reported separately.

5.
Page 13: It is hard to say if there's no toxicity enhancement, since body weight is a crude indicator.A more nuanced statement would be more appropriate.
➢ We changed the words to say that there was no additional body weight loss by the combination (lines 7-8 on page 15).Reviewer #2 -PDAC therapy, fibroblasts (Remarks to the Author):

6.
Nakazawa and colleagues identify and test a novel ADC for the treatment of pancreatic cancer.The manuscript represents a comprehensive preclinical workup of a new therapeutic strategy.The authors identify that a bromodomain and extraterminal (BET) degrader, termed EBET is effective as a payload for an ADC and then choose CEACAM6 as the target for delivery.The authors develop a novel monoclonal antibody #84.7 specific to CEACAM6.#84.7 appears to have selectivity to CEACAM6 expressed on tumor cells and localize marginally to endogenous CAECAM6 on lung epithelial cells in monkeys.The ADC has impressive single agent activity in PDX models of pancreatic cancer.
There are multiple noteworthy results.1. ID of EBET as a therapeutic modality using an organoid screen; 2. Demonstration that EBET alters fibroblast activity (reporter assays) in a co-culture system and reduces induction of a stem-like state in The work provides compelling evidence that 84.7-EBET ADC is an attractive therapeutic that is poised for translation to the clinic.The work is original.
The methods are adequately described and the study encompasses a wide breadth of techniques, results of which in general support the overall conclusions of the study.

Comments:
1.A challenge when developing human specific reagents is the limited toxicity of the construct because normal cells in the mouse are not bound by the therapeutic.The authors provide evidence that 84.7-EBET will be well tolerated in humans (ALI model, in vivo localization in monkeys, no weight loss in mice); however the lack of binding of 84.7 to mouse CEACAM6 complicates interpretation of any toxicity.I suggest that the authors 1. report or determine the MTD of EBET alone in mice; 2. Repeat the ALI study using 84.7-EBET instead of the PNU payload; 3. Determine if 84.7-EBET and KOR-EBET show cytotoxic activity on human PBMCs.
➢ About suggestion 1.We determined MTD of final candidate of EBET in mice and rats (1 mg/kg).This dose is equivalent to 35 mg/kg of ADC when calculated by the amount of loaded payload.

2.
The authors should provide the following clarifications.1.How is the ADC produced, what is the linker between IgG and payload; 2. Is that linker used for all the ADCs in the study; 3. How many EBET moieties are on each 84.7 molecule; 4. What is the concentration of EBET (or payload) delivered in the in vitro studies (e.g.Fig3a,b,c).

Figure 2 -
There is little quantification in these images, making it difficult to determine the level of differences.Is there any way to quantify these results?➢Which images?In case of monkey biodistribution assay (Fig2f), due to the limitations of antibody application in IF staining, we used unfixed fresh-frozen lungs.This made it difficult to unbiasedly prepare a sufficient number of tissue images for quantitation, and only representative images are presented here.Also, at the time of experiment, it was not possible to prepare a sufficient number of monkeys for quantitation.7.Figure2d-How is internalization assessed in this figure?While internalization can occur at 37C, the same signal is generated, so it could be surface binding as well.➢Antibody-treated PDAC cells were resuspended in FBS-containing medium after cell wash, divided into two plates, and incubated at 4 °C or 37 °C for another 2 h.After incubation with secondary antibodies conjugated with fluorescent dye, the cells were analyzed by flow cytometry.Internalization activity was quantitated by comparing mean fluorescence intensity of cells incubated at 4 °C and 37 °C.We modified the figure to see the difference clearly and added the explanation to the manuscript (Fig.2d, lines 17 on page 10 to 1 on page 11, lines 13-17 on page 32).8.What is the membrane permeability of the degrader (e.g.PAMPA)?These larger BRo5 compounds are often more challenging to get into/out of cells.➢EBET compounds have low solubility, and their membrane permeability cannot be measured with PAMPA.However, as mentioned above, the bystander and organoid assays suggest high membrane permeability of EBET compounds.
tumor cells in vitro; 3. Development of a novel mAb specific for CEACAM6 and characterization of the mAb, including showing limited activity of the ADC in an ALI model and in vivo localization studies in monkeys, which helps validate the concept of targeting CEACAM; 4. Demonstration of in vivo efficacy in PDX models that represent classical or basal PDA; 5. Data suggestive of promising activity of the 84.7-EBETADC in combination with anti-PD-1 in an engineered model.

➢
About suggestion 2 and 3.This manuscript is a report of lead CEACAM6-EBET-ADC.After evaluation of the lead ADC, final optimization of linker payload has been conducted especially focusing culture assays with human lung epithelium, hematopoietic progenitor cells and myeloid progenitor cells to improve the tolerability.The final ADC candidate did not show any effect on their viability at 3.3 nM but showed lethal effect in PDAC cells at 0.1 nM.Therefore, we believe we have achieved a certain amount of therapeutic window of the final candidate.The ADC is currently in tolerability testing with dogs and monkeys for clinical trial.Due to our development policies, we are unable to show the structure of the final candidate here but added the data of in vitro efficacy and toxicity and the explanation (Extended Data Fig. 13a-c, lines 2-6 on page 18). 2. The authors should provide the following clarifications.1.How is the ADC produced, what is the linker between IgG and payload; 2. Is that linker used for all the ADCs in the study; 3. How many EBET moieties are on each 84.7 molecule; 4. What is the concentration of EBET (or payload) delivered in the in vitro studies (e.g.Fig 3a,b,c).➢ In this manuscript, we are using cathepsin-B-cleavable GGFG linker.EBET-1593 is released after linker cleavage and self-immolation of aminomethylene moiety.Drug antibody ratio is 4, then the concentration of EBET in the in vitro